Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases which include bovine spongiform encephalopathy (BSE or mad cow disease), Creutzfeldt-Jakob disease (CJD) in humans and scrapie in sheep. A central event in the pathogenesis of TSE diseases is the conversion of normal prion protein (PrP-sen) to an abnormal protease-resistant form (PrP-res). In the past year we have extended our understanding of PrP-res structure and how PrP-res formation can be stimulated and inhibited. Strain-specific conformations of PrP-res. Infrared spectroscopy was used to compare the conformations of PrP-res isolated from hamsters infected with distinct TSE strains. Profound differences in the _-sheet region of the spectrum was observed, providing the first direct evidence that PrP-res types with the same amino acid sequence can have unique conformations. These results support our previous proposals the self-propagation of difference conformers of PrP-res might be a molecular basis of TSE agent strain diversity. Cell-free PrP-res formation under physiological conditions. PrP-res was found to induce the conversion of PrP-sen to PrP-res under physiological pH and salt conditions. The conversion reaction was preceded by the highly selective binding of PrP-sen to PrP-res, which was specifically inhibited either by a synthetic peptide corresponding to PrP residues 109-141 or a monospecific antiserum directed against residues 216-232. These results provide evidence that these residues of PrP-res and/or PrP-sen are critical in the molecular interactions that lead to the induced conversion of PrP-sen to PrP-res. Chaperone supervised PrP-res formation. Since PrP-res formation appears to conformational changes in PrP-sen, we investigated the possibility that chaperone proteins would influence the conversion reaction {3586}. A large panel of chaperone proteins was tested and none induced conversion in the absence of PrP-res. However, in its presence, GroEL and HSP104 promoted conversion. These chaperone proteins cannot be regarded as physiological mediators of the PrP conversion reaction, however, these observations provide a first Aproof-of-principle@ that chaperone proteins can be important cofactors in the PrP conversion process. Inhibition of PrP-res formation by Congo red and analogs. The inhibition of PrP-res formation by Congo red and several of its analogs was compared in scrapie-infected cells and cell-free conversion reactions to learn about the molecular attributes of PrP-res inhibitors. These studies revealed that the coplanarity and/or tortional flexibility of aromatic ring systems is important for Congo red-like inhibitors but that the intersulfonate distance is not a critical parameter.